In order to secure marketing approval for a pharmaceutical product, a manufacturer must submit detailed evidence to the appropriate regulatory authorities to prove that the product is suitable for release on to the market. It is therefore necessary to satisfy regulatory authorities that the product is acceptable for administration to humans and that the particular pharmaceutical composition, which is to be marketed, is free from impurities at the time of release and that it has acceptable storage stability.
Submissions to regulatory authorities must include analytical data which demonstrate that impurities are absent from the active pharmaceutical ingredient (API) at the time of manufacture, or are present at acceptable levels, and that the storage stability of the pharmaceutical composition is acceptable.
The likely impurities in APIs and pharmaceutical compositions include residual quantities of synthetic precursors (intermediates), by-products which arise during the synthesis of the API, residual solvents, isomers of the API (e.g. geometrical isomers, diastereomers or enantiomers), contaminants which are present in materials used in the synthesis of the API or in the preparation of the pharmaceutical composition, and unidentified adventitious substances. Other impurities which may appear during storage include degradants of the API, such as those formed by hydrolysis or oxidation.
The health authorities have very stringent standards and manufacturers must demonstrate that their product is relatively free from impurities or within acceptable limits and that these standards are reproducible for each batch of pharmaceutical product that is produced.
The tests required to demonstrate that the API or pharmaceutical compositions are safe and effective include purity assays, related substances testing, content uniformity testing and dissolution testing. The assay test determines the purity of the test product when compared to a standard of known purity, while the related substances test is used to quantify all the impurities present in the product. The content uniformity test ensures that batches of product like a tablet contain a uniform amount of API and the dissolution test ensures that each batch of product has a consistent dissolution and release of the API.
The technique of choice for the analysis of the API or pharmaceutical compositions (e.g. tablets and capsules) is usually High Performance Liquid Chromatography (HPLC) coupled with a detector. Detectors include UV-visible detectors or mass-spectrometry (MS) detectors. The API and the impurities present, if any, are separated on the HPLC stationary phase and they can be detected and quantified by said detectors.
HPLC is a chromatographic separation technique in which high pressure pumps force the substance or mixture being analysed together with a liquid solvent-mobile phase, also referred to as the eluant—through a separating column containing the stationary phase.
HPLC analysis may be performed in isocratic or gradient mode. In isocratic mode, the mobile phase composition is constant throughout. A gradient HPLC separation is carried out by a gradual change over a period of time in the percentage of the two or more solvents making up the mobile phase. The change in solvent is controlled by a mixer which mixes the solvents to produce the mobile phase prior to its passing through the column.
If a substance interacts strongly with the stationary phase, it remains in the column for a relatively long time, whereas a substance that does not interact with the stationary phase as strongly elutes out of the column sooner. Depending on the strength of interactions, the various constituents of the analyte appear at the end of the separating column at different times, known as retention times, where they can be detected and quantified by means of a suitable detector, such as a UV detector.
Bosentan belongs to a class of highly substituted pyrimidines and is used for the treatment of pulmonary arterial hypertension by blocking the action of endothelin.
Bosentan, having a chemical structure as shown in formula (I), has a molecular weight of 551.615 and its molecular formula is C27H29N5O6S. Bosentan is a white to yellowish white powder and is freely soluble in acetonitrile.

The prior art discloses three process impurities, Ro 47-0005, Ro 47-4056 and Ro 47-9931, obtained during the synthesis of bosentan (EMEA 2005). However, the structures of these impurities were not described. Further, the report also described the formation of three metabolites: Ro 48-5033 (hydroxylation product of the tertiary butyl group), Ro 47-8634 (free phenol metabolite) and Ro 64-1056 (a secondary metabolite, which is free phenol and has a hydroxylated tertiary butyl group).
Several HPLC methods to detect these impurities are reported in the literature, for example: (1) “Evolving Bioanalytical Methods for the Cardiovascular Drug Bosentan”, Chromatographia, vol. 55, pages S115-S119, 2002; and (2) “Determination of an endothelin receptor antagonist in human plasma by narrow-bore liquid chromatography and ionspray tandem mass spectrometry”, J. Chromatography A, vol. 712, pages 75-83, 1995. These publications describe isocratic HPLC methods using mixtures of ammonium acetate and acetonitrile and reverse phase chromatography (RP-18 or RP-8).
However, none of the current HPLC methods are suitable for the detection and quantification of all synthetic intermediates and other related substances that are present in a bosentan sample, particularly a sample synthesised by alternative novel routes, such as the route disclosed in WO 2009/004374 and its priority application IN 1245/MUM/2007. Current methods are also deficient in estimating the total impurities in bosentan and its salts.
Therefore the HPLC methods reported in the prior art are not convenient or suitable for analysing bosentan and its salts as an API, particularly with respect to related substances present in a sample synthesised by the route disclosed in WO 2009/004374 and its priority application IN 1245/MUM/2007.
Consequently, although several HPLC methods have been reported in the literature for the analysis of bosentan and/or its salts and its impurities, there is still a need for an alternative method which avoids the problems associated with the known methods as discussed above.
Studies by the present inventors have lead to the development and validation of a new, efficient, reproducible and simple HPLC method for the analysis of bosentan, particularly with respect to the related substances formed during the synthesis.